Polymerases catalyze the formation of biological polymers. Polymerases are useful for the synthesis of DNA from deoxyribonucleoside triphosphates in the presence of a nucleic acid template and a nucleic acid primer; the synthesis of RNA from ribonucleotides and a DNA or RNA template; DNA replication and repair; and in vitro DNA or RNA amplification.
The 3′ to 5′ exonuclease activity, commonly referred to as “proofreading” activity, is an important characteristic of some DNA polymerases and is present in Pyrococcus species family B polymerases such as Pyrococcus furiosus PolI (referred to herein as “Pfu” and described in U.S. Pat. No. 5,948,663; commercially available from Stratagene, San Diego, Calif.) and Pyrococcus strain GB-D PolI (referred to herein as “Deep Vent®” and described U.S. Pat. No. 5,834,285; commercially available from New England Biolabs, Beverly Mass.). The essential function of the 3′ to 5′ exonuclease is to recognize and cleave a non-base-paired terminus. Enzymes with high exonuclease activity, however, are not commonly used in reactions relying on polymerase activity because they have poor processivity. For example, if used in PCR, it is often in combination with Thermus aquaticus DNA PolI, (Taq), an enzyme with higher processivity but no 3′ to 5′ exonuclease activity, in order to improve the fidelity of the PCR reaction. Improved processivity in polymerases with high 3′ to 5′ exonuclease activity would greatly increase the reliability of reactions relying on the use of polymerases and would eliminate, in some cases, the need for Taq polymerase. Accordingly, a need exists for creating improved polymerases with 3′ to 5′ exonuclease activity.
This invention addresses this and other needs by providing novel compositions with polymerase activity.